Head-to-Head Comparison of Serum Fractionation Techniques

Abstract

Multiple approaches for simplifying the serum proteome have been described. These techniques are generally developed across different laboratories, samples, mass spectrometry platforms, and analysis tools. Hence, comparing the available schemes is impossible from the existing literature because of confounding variables. We describe a head-to-head comparison of several serum fractionation schemes, including N-linked glycopeptide enrichment, cysteinyl-peptide enrichment, magnetic bead separation (C3, C8, and WCX), size fractionation, protein A/G depletion, and immunoaffinity column depletion of abundant serum proteins. Each technique was compared to results obtained from unfractionated human serum. The results show immunoaffinity subtraction is the most effective means for simplifying the serum proteome while maintaining reasonable sample throughput. The reported dataset is publicly available and provides a standard against which emergent technologies can be compared and evaluated for their contribution to serum-based biomarker discovery.

Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*

“…This approach is utilized for validation of preselected candidate markers (4 -6). (ii) Plasma fractionation, which biochemically reduces the complexity of the proteomes, and enables discovery of novel biomarkers (7,8).Targeted MS analysis is dominated by the selected reaction monitoring approach, often in combination with antibodybased enrichment of proteins or peptides and stable isotope labeled standards for quantification (9). This approach benefits from the sensitivity and quantitative capabilities of the triple-quadrupole instruments.…”

“…This approach is utilized for validation of preselected candidate markers (4 -6). (ii) Plasma fractionation, which biochemically reduces the complexity of the proteomes, and enables discovery of novel biomarkers (7,8).…”

Variability of healthy human proteome

“…Однако более детальная оценка этих результатов вызвала сомнения относительно воспроизводимости данных и идентичности белков протеомных паттернов [13][14][15][16][17][18][19]. Главная проблема заключалась в несоответствии дискриминаторных пиков, обнаруженных разными исследовательскими группами даже при использовании одних и тех же методической платформы и биоматериала [20][21][22][23][24][25].…”

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

“…Technological advances such as protein/ antibody chips, depletion of multiple high abundance proteins by affinity columns, and affinity enrichment of targeted protein analytes as well as multidimensional chromatographic fractionation, have all expanded the dynamic range of detection for low abundance proteins by several orders of magnitude in serum or plasma, making it possible to detect the more abundant disease-relevant proteins in these complex biological matrices [63][64][65][66][67][68][69][70][71]. However, plasma and cell-extract based discovery research studies aimed to identify low abundance proteins (e.g.…”